The overall goal of our work is to elucidate the molecular mechanisms of yolk sac blood island organogenesis. These studies will provide new insights into the relationship between blood and endothelial cell development in the first site of blood cell production. To date, reagents to identify the earliest events in yolk sac hematopoiesis have been lacking. We present novel preliminary data that CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo. We have utilized confocal microscopy to identify the site of emergence of the progenitor cells of both primitive and definitive progenitors and have developed a strategy that permits isolation of primitive erythroid progenitor cells (EryP) from embryos or embryonic stem cell-derived embryoid bodies. We propose 3 aims to define the roles of specific molecules in the process of blood island organogenesis: 1. To determine the mechanism of EryP migration and expansion in the proximal yolk sac. We hypothesize that EryP migration and expansion in the proximal yolk sac are VEGF dependent. 2. To define the mechanism of angioblast and extracellular matrix fibronectin (Fn) interaction that results in blood island organogenesis. We hypothesize that blood island formation and remodeling into a capillary bed is a Fn dependent process whereby angioblasts migrate via integrin-Fn interactions to circumscribe the band of primitive erythroblasts in the proximal yolk sac to form intact blood islands. 3. To determine the mechanism of CD41 bright cell cluster formation. We hypothesize that CD41 bright cell clusters require the expression of Runxl and GATA2 for emergence from the endothelial cells located between blood islands and pre-established capillaries (devoid of blood cells) in the distal yolk sac. These studies will define molecular pathways required for blood island development and remodeling.